Abstract Background Mizoribine (MZR) has been developed as an immunosuppressive agent in Japan, but it shows less potent immunosuppressive effects at doses up to 3 mg/kg/d. In this study, we investigated whether high-dose MZR (6 mg/kg/d) was effective for ABO-incompatible (ABO-i) living donor kidney transplantation (LKT) using treatment with anti-CD25 and anti-CD20 monoclonal antibodies without splenectomy. Methods Since 2007, we encountered 24 cases of ABO-i LKT using anti-CD20 and anti-CD25 monoclonal antibody without splenectomy. The pretransplant immunosuppressive regimen consisted of two doses of anti-CD20 antibody, mycophenolate mofetil (MMF), prednisolone, a calcineurin inhibitor (cyclosporine 7 mg/kg or tacrolimus 0.2 mg/kg and two doses of anti-CD25 antibody. Antibody removal by plasmapheresis was performed before LKT up to several times according to the antibody titer. The posttransplant regimen consisted of high-dose mizoribine (6 mg/kg/d) instead of MMF (MZR group, n = 12). Results The 1-year graft survival rates for the MZR and MMF groups were both 100%. The rejection rate in the MZR group (eight %) was not significantly higher than that in the MMF group (seventeen %) Serum creatinine level was not significantly different between the two groups. In the MZR group 6 (50%) patients developed CMV antigenemia-positivity versus 11 (92%) in the MMF group ( P < .05). The number of patients who developed CMV disease was 0 in the MZR group and 1 (8%) in the MMF group. The number of patients treated with ganciclovir was 0% and 8%, respectively (not significant). Conclusions We obtain good clinical results with high-dose MZR in ABO-i LKT using anti-CD20 and anti-CD25 antibody treatment without splenectomy.